There is a continuing need for improved expression vectors exhibiting high levels of expression. In particular, expression vectors for use in mammalian cell lines are of increasing importance both for the industrial production of desired polypeptides and for the development of therapies for genetic disorders.
There are many known examples of characterised structural genes, which together with appropriate control sequences may be inserted into suitable vectors and used to transform host cells. A significant problem with the integration of such a structural gene and control regions into the genome of a mammalian cell is that expression has been shown to be highly dependent upon the position of the inserted sequence in the genome. This results in a wide variation in the expression level and only very rarely in a high expression level. The problem of integration site dependence is solved by the present invention and arises from the discovery of specific sequences referred to herein as dominant control regions (DCRs) derived from immunoglobulin genes which have the property of conferring a cell-type restricted, integration site independent, copy number dependent expression characteristic on a linked gene system.
Two mammalian gene systems have been shown to possess DCRS, namely the .beta.-like globin genes (Grosveld, F. G. et al, Cell, 51, (1987), 975; International patent application PCT/GB 88/00655), and the human CD2 T-cell marker gene (Lang, G., et al, EMBO J, 7, (1988), 1675; International patent application PCT/GB 88/00655).
When a mammalian gene such as .beta.-globin containing all the usual control regions is introduced into transgenic mice, the gene is not expressed at the same level as the mouse .beta.-globin gene and exhibits integration site position effects. This is characterized by a highly variable expression of the transgene that is not correlated with the copy number of the injected gene in the mouse genome. The same phenomenon has been observed in almost all the genes that have been studied in transgenic mice (Palmiter et al, Ann. Rev. Genet., (1986), 20, 465-499). Moreover, the level of expression of each injected gene in the case of .beta.-globin is, at best, an order of magnitude below that of the endogenous mouse gene (Magram et al., Nature, (1985), 3, 338-340; Townes et al., EMBO J., (1985), 4 1715-1723; Kollias et al, Cell, (1986), 46, 89-94). A similar problem is observed when the .beta.-globin or other genes are introduced into cultured cells by transfection or retroviral infection. This poses a big problem when considering gene therapy by gene addition in stem cells. It is also a major problem for the expression of recombinant DNA products in cultured cells. Extensive screening for highly producing clones is necessary to identify cell-lines in which the vector is optimally expressed and selection for vector amplification or use of multicopy viral vectors is generally required to achieve expression levels comparable to those of the naturally occurring genes, such as for example .beta.-globin genes in erythroleukaemic cell-lines.
In a chromosome, the genetic material is packaged into a DNA/protein complex called chromatin, one effect of which may be to limit the availability of DNA for functional purposes. It has been established that many gene systems (including the .beta.-globin system) possess so-called DNaseI hypersensitive sites. Such sites represent putative regulatory regions, where the normal chromatin structure is altered, for instance by interaction with regulatory proteins or to allow such interaction.
Regions flanking the .beta.-like globin gene locus which contain a number of "super" hypersensitive sites have been identified. These sites are more sensitive to DNase I digestion in nuclei than the sites found in and around the individual genes when they are expressed (Tuan et al PNAS USA, (1985), 2, 6384-6388; Groudine et al, PNAS USA, (1983), 80, 7551-7555). In addition, they are erythroid cell specific and they are present when any one of the globin genes is expressed.
Tuan et al describe the broad mapping of four major DNase I hypersensitive sites in the 5' boundary area of the ".beta.-like" globin gene. The authors note that certain sequence features of these sites are also found in many transcriptional enhancers and suggest that the sites might also possess enhancer functions and be recognised by erythroid specific cellular factors.
It has been discovered that the complete .beta.-globin gene with intact 5' and 3' boundary regions does not exhibit an integration site position dependence (see copending International patent application PCT/GB 88/00655). The regions of the locus responsible for this significant characteristic have been determined and shown to be associated with DNase I super hypersensitive sites. These dominant control regions are quite distinct from enhancers, exhibiting properties such as-integration site independence not exhibited by the known enhancers. The dominant control region used in conjunction with the known promoter/enhancer elements reconstitute the full transcription rate of the natural gene. immunoglobulin genes have been extensively studied in order to identify sequences regulating gene expression. An immunoglobulin molecule consists of two identical heavy polypeptide chains and two identical light polypeptide chains. The light chains may be either of the .kappa. or .lambda. type. The genes encoding the heavy chain, the .kappa. light chain and the .lambda. light chain are each located on separate chromosomes in the mouse and man.
Unlike most genes which are transcribed from continuous genomic DNA sequences, immunoglobulin genes are assembled from gene segments which may be widely separated in the germ line.
Functionally, heavy chain genes are formed by recombination of three genomic segments encoding the variable (V), diversity (D) and joining (J)/constant (C) regions of the molecule (FIG. 1). Functional light chain genes are formed by joining of two segments, one encoding the V region and the other the J/C region. Both the heavy chain and .kappa. light chain loci contain many V gene segments (estimates vary between 100s and 1000s) estimated to span well over 1000 kb (FIG. 1). The .lambda. locus is, by contrast, much smaller and has recently been shown to span approximately 300 kb on chromosome 16 in the mouse. It consists of four joining/constant region gene segments and two variable gene segments (FIG. 1). Recombination resulting in functional genes occurs predominantly between V.sub.1 and either J.sub.1 /C.sub.1 or J.sub.3 /C.sub.3 elements or between V.sub.2 and J.sub.2 /C.sub.2 elements (J.sub.4 /C.sub.4 is a pseudogene) although recombinations between V.sub.2 and J.sub.3 /C.sub.3 or J.sub.1 /C.sub.1 are seen very rarely.
Control of transcription of both rearranged heavy and .kappa. light chain genes depends both on the activity of a tissue specific promoter upstream of the V region (Mason, J. O. et al, Cell, (1985), 41, 479; Bergman, Y. et al, PNAS USA, (1984), 81, 7041) and a tissue specific enhancer located in the J-C intron (Gillies, S. D. et al, Cell, (1983), 33, 717; Banerji, J. et al, Cell, (1983), 3, 729; Picard, D. et al, Nature, 307, 80). These elements act synergistically (Garcia, J. V. et al, Nature, (1986), 3, 383). Recently a second B-cell specific enhancer has been identified in the .kappa. light chain locus (Meyer, K. B. et al, EMBO J., (1989), 8, No. 7,1959-1964). This further enhancer is located 9 kb downstream of C.sub..kappa..
More recently Bich-Thuy and Queen (1989 NAR 17:5307) described an enhancer activity immediately downstream of the rearranged .lambda..sub.1 gene. Sequences downstream of the .lambda..sub.1 gene increased expression of a chloramphenicol acetyl transferase (CAT) reporter gene linked to the .lambda..sub.1 promoter in a myeloma cell line which made lambda light chains (J558L) but not in two myeloma cell lines which make .kappa. light chains.
This enhancer activity differs in several ways from that of the heavy chain and .kappa. light chain enhancers. Firstly, it displayed a marked orientation preference. Secondly, it consists of several segments which can independently stimulate transcription and which are spread over about 4 kb of DNA immediately downstream of the .lambda..sub.1 coding sequence. Thirdly, it is apparently only expressed in .lambda. chain producing myeloma cells and not in cells producing .kappa. light chains.
Spandidos and Anderson (1984 FEBS Lett. 175: 152) describe an 8 kb fragment (containing 2 constant region gene segments) from the human .lambda. locus which increases the number of G418 resistant colonies obtained after transfection of myeloma cells with a plasmid containing the 8 kb fragment linked to the aminoglycoside-phosphotransferase (aph) gene under the control of an .epsilon.-globin promoter. Levels of aph specific mRNA were reported to be increased following transient transfection of this construct when compared with a construct lacking the 8 kb sequence. However, no controls were presented for transfection efficiency in the transient assays and no further reports relating to enhancer activity in the human I locus have been published.
DNA fragments carrying rearranged heavy or .kappa. light chain genes are expressed when transfected into lymphoid cells although generally at least 10 times less efficiently than endogenous immunoglobulin genes (Oi, V. T. et al, PNAS USA, (1983), 80, 825; Neuberger, M. S., EMBO J., (1983), 2, 1373). Expression of a similar DNA fragment containing a rearranged .lambda.1 gene however is not detectable in transfected lymphoid cells and expression is only observed when an SV40 enhancer is added (Picard, D. et al, Nature, (1984), 307, 80; Cone, R. D. et al, Science, (1987), 236, 954). This is apparently due to the absence of a functional enhancer such as that present in the J-C intron of the .kappa. gene.
Although promoter and enhancer elements associated with .kappa. and heavy chain genes are sufficient to confer lymphoid cell specific expression of antibody genes, the level of expression is reduced compared with expression of endogenous antibody genes and genes are subject to position effects such that no clear relation exists between the level of expression and the copy number of the introduced gene (Oi, V. T. et al, PNAS USA, (1983), 80, 825; Neuberger, M. S., EMBO J., (1983), A, 1373). Similar results are obtained when .kappa. and heavy chain genes are introduced into transgenic mice (Storb U., et al, Ann. Rev. Immunol., 5, (1987), 151).
Sequences required for full regulation of antibody gene expression are therefore lacking from constructs used to date in expression studies (Grosschedl, R. et al, Cell, (1985), 55, 645).
We have now identified super hypersensitive sites in the rearranged .lambda..sub.1 mouse immunoglobulin gene outside of the region previously suggested to be involved in expression of the .lambda..sub.1 gene. The strong implication of our finding is that immunoglobulin gene expression shares a common feature with .beta.-globin and CD2 expression, namely the existence of dominant control regions.
Many active genes have hypersensitive sites associated with their promoters. For example, a tissue specific DNaseI hypersensitive site is observed 300 bp upstream from the start of the coding region of the rearranged .kappa. immunoglobulin gene (Chung, S-Y et al, PNAS USA, 80, 2427). No such site was identified in the rearranged .lambda..sub.1 gene which was studied in the present work nor was any site identified in the J-C intron where hypersensitive sites associated with immunoglobulin enhancers have been identified in both the heavy chain (Mills, F. et al., (1984), Nature, 306, 807) and light chain genes (Chung, S-Y et al, PNAS USA, 80, 2427; Parslow, T. G. et al, NAR, (1983), 11, 4775); Weischet et al, NAR, (1982), 10, 3627).
The hypersensitive sites characteristic of the dominant control regions of the .beta.-globin locus identified by Grosveld et al. (Grosveld, F. G. et al, Cell, (1987), 51, 957-985) are referred to as "super hypersensitive". The designation "super" hypersensitive refers to the fact that these sites are much more sensitive to DNaseI that the "normal" hypersensitive site associated with the .beta.-globin promoter (Groudine, M. et al, PNAS USA, (1983), 80, 7551). In the mouse .lambda. locus, promoter hypersensitive sites are not detectable and the only DNaseI hypersensitive sites which are seen, map at larger distances from the gene.
The hypersensitive sites in the mouse .lambda. locus indicate locations of sequences which, according to the present invention confer dominant control of immunoglobulin genes. On the basis of this mapping, DNA sequences flanking the rearranged gene in J558L have been cloned.
The present invention is applicable to the production of transgenic animals and the techniques for producing such are now widely known. For a review, see Jaenisch, Science, (1988), 240, 1468-1474.
The present invention provides a solution to the problem of integration site dependence of expression making possible the insertion of functionally active gene systems into mammalian genomes both in vitro and in vivo. Specifically, the present invention provides a vector capable of expressing homologous and heterologous genes after their introduction into immunoglobulin producing cells, whether in vivo or in vitro, in an integration-site independent cell specific manner.
We have found also that certain of the hypersensitive sites in the mouse .lambda. locus correspond to new useful .lambda. immunoglobulin enhancer elements. Thus the present invention further provides these new enhancer elements and vectors containing these elements, useful for the expression of heterologous genes.